Effect of Lyophilised Sumac Extract on the Microbiological, Physicochemical, and Antioxidant Properties of Fresh Carrot Juice

Abstract

The lyophilised sumac (Rhus coriaria L) extract (LSE), in amounts of 0.25, 0.5, 1.0, 1.25, and 1.5 g/100 ml of juice, was incorporated into carrot juice, and its properties were assessed after 24, 48, and 72 h. A product without the lyophilised sumac extract served as the control. The highest supplementation level enhanced the physicochemical characteristics of carrot juice, increasing carotenoid and polyphenolic contents by 22% and 70% on the first day. The LSE significantly boosted antioxidant activity, yielding over a tenfold increase, while reducing capacity was elevated more than sevenfold. LC-MS analysis confirmed the presence of bioactive compounds, such as chalcones, flavonols, flavones, and phenolic acids, further validating the extract’s functional potential. Acidity and redness exhibited a proportional increase with the rising concentrations of the additive used. Additionally, microbial growth, including aerobic mesophiles, yeasts, and moulds, was markedly suppressed. After 72 h, the total count of aerobic microorganisms and yeasts/mould was reduced by 5.64 log and 4.94 log, respectively, compared to the control. The lyophilised sumac extract, rich in valuable bioactive compounds with antioxidant properties, effectively preserved freshly pressed carrot juice, mitigating spoilage and extending its shelf life. This form of sumac serves as a sustainable beverage additive, minimises food waste, and aligns with clean-label trends.

1. Introduction

The food industry is increasingly trying to implement novel ways of preserving products, which can also help to modify their functionality. Not-from-concentrate (NFC) juices align with this trend by maintaining their original freshness and nutritional integrity, offering consumers a more natural and minimally processed option [1,2]. Their composition, rich in bioactive compounds, such as vitamins, polyphenols, and flavonoids, contributes to enhanced health benefits while supporting the demand for sustainable and transparent food production [3,4]. Research continues to explore antimicrobial and antioxidant properties of herbs and spices, which gain recognition not only for their role in flavour enhancement but also for their broader implications in health and food security [5,6]. The use of a properly balanced diet and herbal preparations as a form of preventive therapy is an important element of the sustainable development strategy in health and environmental protection. Supporting natural methods of treatment can contribute to limiting the excessive use of synthetic pharmaceutical substances, which directly contributes to the reduction of pollutant emissions and the burden on water and soil ecosystems [7,8,9]. Additionally, research indicates that bioactive plant compounds, such as polyphenols and flavonoids, can be an effective alternative to synthetic antioxidants, supporting the body’s homeostasis and reducing the risk of lifestyle diseases [10,11].

Consumers demand natural additive-free (‘clean label’) beverages; hence, incorporating plant extracts with health benefits is a promising approach. A spice prepared from the fruit of a shrubby plant belonging to the genus Rhus coriaria L. exhibits strong health-promoting properties, as it contains natural substances that are powerful antioxidants (polyphenols: mainly flavonoids, vitamin C) [12,13,14,15].

In recent years, there has been increased interest in polyphenolic compounds due to their antiatherosclerotic, anti-cancer, and anti-inflammatory effects. They are contained in many dietary supplements, although they can also be easily incorporated into the diet from foods, such as fruit and vegetables. A particularly important role in the prevention of lifestyle diseases is attributed to their preparations, with the largest share of juices. However, the type and variety of raw materials, processing, and storage parameters may be responsible for a significant variation in the amount of polyphenolic compounds found in beverages. Moreover, the competition in the juice market forces producers to take special care of the quality of the products they offer to meet the demands expressed by consumers who are paying increasing attention to a healthy lifestyle. When choosing food, they are guided not only by the price or brand but also by health-promoting properties [16,17,18].

Sumac is a source of valuable minerals, such as potassium, magnesium, calcium, and omega-3 acids. Furthermore, extracts of Rhus coriaria fruit are effective in inhibiting the growth of undesirable microorganisms, including foodborne bacteria (Bacillus cereus, Staphylococcus aureus, Escherichia coli, Yersinia enterocolitica, Shigella dysenteriae, Salmonella enteritidis) [19,20,21,22]. Experiments have shown that sumac can be considered as a natural agent increasing the shelf life of such products as chicken wings [23], ground sheep meat [24], ground beef [25], fillets of rainbow trout [26], silver carp [27], tomato paste [28], milk [28], cheese [29], and beverages [16,30]. The antioxidant activity of sumac extracts in stabilising peanut oil against oxidative degradation was reported by Özcan [31]. Moreover, tannin-rich sumac extracts have been shown to improve the oxidative stability of dairy and meat products [32].

In general, extracts are a convenient and effective form of food enrichment, providing concentrated bioactive ingredients [33,34,35]. The lyophilisation (freeze-drying) process not only increases their concentration but also improves their chemical and physical stability, which plays a key role in maintaining their effectiveness in food products. By removing water, the risk of degradation reactions, such as hydrolysis and oxidation, is significantly reduced. Additionally, the low temperature of the lyophilisation process minimises thermal degradation, thereby preserving the structural integrity and biological activity of heat-sensitive compounds. It also prevents textural changes, such as clumping or phase separation, during storage. Furthermore, the porous structure of lyophilised extracts ensures better control over the release of active ingredients into the food matrix, which can improve their bioavailability. Lyophilised extracts are characterised by better solubility, making their presence in the final product almost invisible, which is particularly important in terms of sensory acceptance [36,37,38,39]. Several studies have demonstrated that lyophilised extracts of Rhus coriaria (sumac) exhibit higher antioxidant activity and retain a greater concentration of phenolic compounds than extracts obtained using conventional drying techniques. For instance, Kosar et al. [40] showed that lyophilised sumac extracts contained significantly more gallic acid and flavonoid glycosides than air-dried equivalents, with markedly higher radical scavenging activity. Similarly, Bursal and Köksal [41] reported that freeze-dried water and ethanol extracts of sumac exhibited stronger DPPH and reducing power than those processed in thermal conditions [42]. Lyophilised sumac extracts have demonstrated increased antioxidant activity, possibly as a result of reduced oxidative and thermal degradation during the drying process [33]. These findings underline the value of lyophilisation as a superior method for preserving the functional and nutritional quality of plant-derived extracts, including sumac, in food applications.

The aim of the study was to evaluate the effect of the addition of a lyophilised sumac extract (LSE) to carrot juice on the physicochemical properties, antioxidant activity, bioactive content, and microbiological safety of the product.

2. Materials and Methods

Dried and ground sumac fruits and carrots (Nerac variety) with cylindrical roots and no visible mechanical damage were purchased from a local supermarket (Lublin, Poland).

2.1. Preparation of Lyophilised Sumac Extract (LSE)

The LSE was prepared using dried and ground Rhus coriaria L. fruits and distilled water at a 1:5 (w/v) ratio during the extraction process. The mixture was placed in a shaking temperature-controlled water bath (SWB 8N Laboplay, Bytom, Poland) at 40 °C for 3 h. The extract was filtered using Whatman No. 1. The aqueous extract obtained in this way was then lyophilised (Free Zone 12 lyophiliser, Labconco Corporation, Kansas City, MO, USA) in appropriate conditions, placed in sealed bottles, and stored at 4 °C.

2.2. Preparation of Carrot Juice with Lyophilised Sumac Extract

Fresh carrot roots with ends removed were washed thoroughly and peeled with a sharp knife. The juice was pressed using a slow juicer (Sana EUJ-707, Omega Products, České Budějovice, Czech Republic). The lyophilised sumac extract was added to the juice in the amounts of 0.25, 0.5, 1.0, 1.25, and 1.5 g/100 mL juice and stored refrigerated (6 °C) for three days in glass packaging, with daily quality analyses of the products. Juice without the sumac addition was the control sample.

2.3. Microbiological Tests

Microbiological tests were carried out with the plate method using cultures in a laminar chamber with a CRUMA 670FL UV lamp (El Prat de Llobregat, Barcelona, Spain) according to the current methodological standards [43,44]]. In the study material, the total aerobic bacterial counts were determined on nutrient agar called Plate count agar (PCA), and the yeast and mould counts were evaluated using agar with dichloran and 18% glycerol (DG18) (Biomaxima, Lublin, Poland). Prior to the cultures, serial dilutions of the samples (from 10⁻¹ to 10⁻⁵) were prepared by transferring 1 mL of juice into sterile falcones containing 9 mL of peptone water. Then, 0.1 millilitres of each dilution was spread on the surface of the plates with the prepared medium. Aerobic mesophiles were incubated at 30 °C for 72 h, and yeasts and moulds were incubated at 25 °C for 5 days in a CLN 115 SMART incubator (POL-ECO, Wodzisław Śląski, Poland).

2.4. Physicochemical Properties of Juice

2.4.1. pH

The pH value was determined with a glass electrode attached to a pH meter (model number 780 pH Meter, Metrohm, Herisau, Switzerland) with a temperature probe after calibration (buffer solutions pH 4 and 7).

2.4.2. Total Soluble Solids (TSS)

The content of total soluble solids (TSS) in the juice was determined with a digital refractometer LLG-uniREFRACTO (Meckenheim, Germany), and the results are expressed in °Brix.

2.4.3. Determination of CIE Lab Colour Parameters

The CIE Lab colour parameters (L*, a*, b*) of the juice were measured using a reflectance spectrophotometer 3Color SF80 (TRI-COLOR, Narama, Poland) with an angle of observation of 10° and the D65° illuminant.

2.5. Bioactive Compounds

2.5.1. Determination of Vitamin C

Vitamin C in the tested carrot juices was determined with the iodometric method [45]. It is based on the reduction of iodine by ascorbic acid and the formation of an addition compound of iodine with blue-coloured starch after all ascorbic acid had reacted. The juice sample was titrated in the presence of a 1% starch solution with a standard iodine solution until a dark blue colour appeared. The titre of the iodine solution was determined using the titration of 1 cm³ of an L-ascorbic acid solution of known concentration. The results are expressed in milligrams per 100 g of juice.

2.5.2. Total Carotenoid Content (TCC)

The total carotenoid content (TCC) was determined according to the method described by González-Casado et al. [46]. The carrot juice (1 g) was mixed with acetone (with the addition of 0.2% butylhydroxytoluene), ethanol, and hexane in a ratio of 1:1:2 (50 mL). The colour absorbance of the hexane phase was measured at 450 nm with a UV/Vis Helios Omega 3 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The results are expressed in milligrams per 100 g of juice.

2.5.3. Total Phenolic Content (TPC)

The total phenolic content was determined with the Folin–Ciocalteu method [47] after 80% methanol extraction from the carrot juice. The samples were extracted in conical flasks, with agitation in orbital shaker S-3.02.20M (ELMI Ltd., Ryga, Łotwa), (200 rpm, for 30 min). The extract (0.1 mL each) was mixed with distilled water (2 mL), Folin–Ciocalteu phenol reagent (0.2 mL), and 20% sodium carbonate (2 mL). It was allowed to stand at room temperature (40 min), the absorbance was measured at 765 nm using a UV/Vis Helios Omega 3 spectrophotometer (Thermo Scientific, Waltham, MA, USA), and the result was expressed as milligrams of gallic acid per 100 mL sample.

2.6. Antioxidant Activity

2.6.1. DPPH Radical Scavenging Activity Assay (DPPH)

The DPPH assay was conducted according to Brand-Williams et al. [48] with slight modification. The reaction mixture consisted of 100 µL of the extract and 4 mL of a 0.1 mM DPPH solution freshly prepared prior to the analysis. The sample was stored at room temperature for 30 min, and then the absorbance was measured at λ = 515 nm on a Cary 50 spectrophotometer (Varian, Palo Alto, CA, USA). A calibration curve was used to determine the DPPH radical scavenging values, and the results are expressed as mmol Trolox equivalent (TE)/100 mL.

2.6.2. ABTS Radical Scavenging Activity Assay (ABTS)

The ABTS radical scavenging activity of the samples was measured using the ABTS method [49]. A 7 mM ABTS aqueous solution was mixed with 2.45 mM potassium persulfate and allowed to react in the dark at room temperature for 12–16 h to generate the ABTS radical cation. Prior to the analysis, the ABTS solution was diluted with ethanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. For the assay, 3 mL of the ABTS working solution was mixed with 100 µL of the test sample. The decrease in absorbance was measured at 734 nm using a Cary 50 spectrophotometer (Varian, Palo Alto, CA, USA). A calibration curve was used to determine the ABTS radical scavenging values, and the results are expressed as mmol Trolox equivalent (TE)/100 mL.

2.6.3. Ferric Reducing Antioxidant Power Assay (FRAP)

The FRAP assay was performed following the method described by Benzie and Strain [50]. The FRAP working reagent was freshly prepared by mixing 2.5 mL of a 10 mmol/L TPTZ solution, 2.5 mL of 20 mmol/L FeCl₃, and 25 mL of 0.3 mol/L acetate buffer (pH 3.6), followed by incubation at 37 °C. For the assay, 2.8 mL of the FRAP reagent was combined with 200 µL of the test sample. The reaction mixture was then incubated at 37 °C for 30 min, after which the absorbance was measured at 593 nm on a Cary 50 spectrophotometer (Varian, Palo Alto, CA, USA). A calibration curve was used to calculate the FRAP values, and the results are expressed as mmol Trolox equivalents/100 mL.

2.7. Phenolic Compound Profile—LC-MS

The individual phenolic compounds in the samples were determined using the liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method. The method for the determination of compounds used by Staszowska-Karkut et al. [51] with modifications was employed. The analyses were performed using an Agilent Technologies 1290 series liquid chromatograph coupled to an Agilent Technologies 6530 Q-TOF LC/MS high-resolution mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation was carried out on a C18 column, 2.1 × 10 mm, grain 1.8 µm. The mobile phase consisted of 0.1% formic acid in acetonitrile (A) and 0.1% formic acid water (B) with the elution gradient and flow of 0.4 mL/min. Mass spectra were obtained in the mass range of 100–2000 Da with a scan time of 1.0 s operated in the positive (ESI+) and negative (ESI−) ionisation modes. Data were collected using the “MassHunter Acquisition” and “MassHunter Qualitative Analysis” software version B.10.00 Agilent Technologies, Inc. (Santa Clara, CA, USA). The “Personal Compound Database (PCD) and Library Software” version B.08.00 SP1 system was used to interrogate the database and library directly, where the identification of the compound was carried out using the Find-by-Formula (FBF) algorithm.

2.8. Statistical Data Analysis

The data from microbiological (n = 4) and physicochemical analyses and the determination of bioactive compounds and antioxidant activity (n = 3) of juice samples were analysed using Statistica software version 10 (Tulsa, OK, USA). One-way analysis of variance was used. The addition of LSE was the factor differentiating the groups. Tukey’s test was used to compare means. Significance was established at p < 0.05.

3. Results and Discussion

3.1. Microbiological Analyses

The detailed results of the microbiological analyses are shown in Figure 1 and Figure 2. The mesophilic aerobic bacterial counts in the juice without the additive were on average 6.36 log CFU/mL, 7.43 log CFU/mL, and 9.61 log CFU/mL after 24, 48, and 72 h of storage, respectively. In contrast, the microbial counts in the LSE-enriched samples declined with the increasing additive concentrations, ranging from 5.49 to 4.23 log CFU/mL after 24 h (for 0.25 and 1.5 g LSE/100 mL juice, respectively), from 6.78 to 4.50 log CFU/mL after 48 h, and from 7.58 to 3.97 log CFU/mL after 72 h. Evidently, the increase in the concentration of the extract in the juice resulted in greater inhibition of the growth of microorganisms. The results showed that the 1.5 g addition of lyophilised sumac on 100 mL juice reduced the total aerobic microbial count by more than 58% (5.64 log CFU/mL), compared to the control sample, when stored under refrigeration for 72 h. This yielded a juice that, according to the Codex Standards, was within the standard that defines it as a drinkable product (not exceeding 4 log CFU/mL) (Figure 1).

Zou and Jiang [45] analysed non-pasteurised carrot juice and showed that the total microbial count was 3.97 log CFU/mL in fresh juice analysed immediately after squeezing. Compared to their results, the values obtained in our study were significantly higher, which may be due to the different composition of the initial microflora or different experimental conditions.

Figure 2 shows the results of yeast and mould counts in the control and LSE-supplemented carrot juice during storage for 72 h. The values in the control samples ranged from 4.38 log CFU/mL (after 24 h) to 5.54 log CFU/mL (after 72 h). The addition of the LSE significantly inhibited the growth of these microorganisms already after the first day of juice storage. The number of yeasts and moulds in the experimental samples after this time was on average between 3.49 log CFU/mL (0.25 g additive) and 2.00 log CFU/mL (1.5 g additive). The further storage of the LSE-enriched juice additionally slowed down the proliferation of fungi. After 48 h, the highest average reduction in yeasts and moulds (by 2.93 log CFU/mL, compared to the control) was observed in the sample supplemented with 1.5 g/100 mL of the lyophilised sumac extract. In contrast, after 72 h of storage, the number of these microorganisms was even lower, i.e., on average 0.61 log CFU/mL. In this case, the addition of the lyophilised sumac extract reduced the growth of yeasts and moulds by nearly 89%, compared to the control sample.

Literature reports show varying levels of mould contamination in fresh vegetable juices. For example, Sokołowska et al. [52] detected the highest numbers of yeasts and moulds, amounting to 10³–10⁵ CFU/mL and 10²–10⁴ CFU/mL, respectively, in samples analysed in their study.

The statistical tests of microbiological results showed that the addition of LSE had a statistically significant effect on the changes in the microbial count in the juice (mesophilic aerobic bacteria as well as yeasts and moulds), compared to the control sample (

Table 1. ANOVA analysis of the LSE addition effect on the properties of carrot juice.

Parameters	LSE Addition (g/100 mL)
	0.25	0.5	1.0	1.25	1.5
Mesophilic aerobic bacterial counts	+	+	+	+	+
Number of yeasts and moulds	+	+	+	+	+
pH	+	+	+	+	+
TSS			+	+	+
L*	+	+	+	+	+
a*	+	+	+	+	+
b*	+	+	+	+	+
Vitamin C					+
TCC		+	+	+	+
TPC		+	+	+	+
DPPH		+	+	+	+
ABTS			+	+	+
FRAP			+	+	+

+—statistically significant differences compared to the juice without LSE.

). Flavonoids present in sumac exhibit antibacterial activity through damage to cytoplasmic membranes and inhibition of nucleic acid synthesis and energy metabolism [53]. Biofilm formation inhibition may be related to the ability of plant extracts/flavonoid components to inactivate microbial adhesins and enzymes, altering the bacterial surface, thus disrupting and impairing cell-substrate interactions, the attachment phase, and normal biofilm development. The aggregate effect of flavonols on whole bacterial cells may also determine the preferred interaction of bacterial cells with each other rather than with the surface [54].

To date, most studies showing the positive effects of sumac on the microbiological quality of food products have been carried out on raw meat and fish [22,27]. Our findings suggest the possibility of using Rhus coriaria L. fruit lyophilisates as a new source of natural antimicrobial substances for juice manufacturing. Terpene hydrocarbons, oxygenated terpenes, famesyl acetone, hexahydrofamesyl acetone, aliphatic aldehydes, and most importantly, tannins are considered to be the active constituents present in this plant species. The latter compounds play the most important role in the inhibition of extracellular enzymes and oxidative phosphorylation, which results in disruption of the bacterial membrane and nutrient deprivation in substrates [21,55,56,57].

3.2. Physicochemical Properties

An important indicator of the quality and freshness of juices is their pH value, which is closely related to the biochemical reactions occurring during storage. The analysed juices were characterised by decreasing pH values after the addition of LSE. The products showed a pH value ranging from 4.26 in the sample with the 1.5 g addition to 6.47 in the control sample after 24 h. At 48 h, the pH of the samples with the LSE addition oscillated between 4.15 and 5.38, while the average value of the parameter in the juice without the supplement was 6.23. The decrease in pH during storage may be related to the proliferation of microflora utilising sugars for the production of acids. It should be mentioned that the control samples and those enriched with 0.25 g and 0.5 g of LSE were spoiled after 72 h. They had an unpleasant odour and a mucilaginous consistency and were therefore excluded from further testing for safety reasons. The pH of the products supplemented with lyophilised sumac extract after 72 h ranged from 4.30 to 4.52 (

Table 2. pH values and total soluble solids (TSS) in carrot juice stored for 24, 48, and 72 h.

LSE Addition (g/100 mL)	Storage Time (h)
	24	48	72
pH
0 (control)	6.47 ± 0.08	6.23 ± 0.09	JS
0.25	5.61 ± 0.00	5.38 ± 0.14	JS
0.5	5.14 ± 0.00	4.94 ± 0.05	JS
1.0	4.59 ± 0.01	4.37 ± 0.08	4.52 ± 0.04
1.25	4.43 ± 0.00	4.33 ± 0.05	4.38 ± 0.00
1.5	4.26 ± 0.01	4.15 ± 0.07	4.30 ± 0.01
TSS (°Brix)
0 (control)	10.03 ± 0.15	10.47 ± 0.15	JS
0.25	10.20 ± 0.10	10.80 ± 0.10	JS
0.5	10.73 ± 0.06	11.03 ± 0.15	JS
1.0	11.67 ± 0.21	12.03 ± 0.21	11.17 ± 0.06
1.25	11.40 ± 0.36	11.47 ± 0.38	10.77 ± 0.21
1.5	11.07 ± 0.12	11.50 ± 0.10	11.97 ± 0.15

The results are expressed as a mean (n = 3) ± standard deviation from three independent experiments. JS—juice spoiled.

).

A study conducted by Wang et al. [58] confirmed the acidifying nature of sumac; when added in varying amounts to Cheddar cheese, it reduced the pH of samples relative to control products. Various sumac genotypes contain many organic acids (malic, citric, fumaric, tannic, gallic, and ascorbic acids) [31,59] and can influence the values of this parameter to a greater or lesser extent. The low-acid nature of carrot juice makes it more susceptible to spoilage, which can be prevented by acidification. Commercially available juices are usually enriched with ascorbic acid or supplemented with other acidic additives, such as apple juice. Such measures prevent oxidative processes, thereby improving the dietary qualities of the final product.

The total soluble solids (TSS) in the analysed carrot juices ranged from 10.03 to 12.03 °Brix (Table 2). The TSS level of 10.03 °Brix in the control juice sample was close to the values reported by Rodriguez et al. [60] and higher by about 2.5° than in the carrot juice extract analysed by Jabbar et al. [61] and Umair et al. [62]. The statistical tests showed that the addition of LSE at 1.0, 1.25, and 1.5 g/100 mL resulted in a statistically significant increase in the content of total soluble solids, compared to the control sample (Table 1). The slight increase in the extract content in the juices during storage may have been caused by the further dissolution of its components in the juice matrix and by the slow diffusion of compounds from the lyophilisate microstructure.

As consumers become more interested in highly nutritious foods, there is more competition, and juice producers need to design higher quality products to stand out in the competitive market. The first human-perceived indicator of the quality of food is its colour. Measuring colour makes it possible to design new products that are attractive to buyers.

Table 3. Colour indices of carrot juice stored for 24, 48, and 72 h.

LSE Addition (g/100 mL)	Storage Time (h)	Colour Index
		L*	a*	b*
0 (control)	24	37.63 ± 0.10	11.67 ± 0.03	16.53 ± 0.07
0.25		39.15 ± 0.03	15.59 ± 0.07	20.70 ± 0.08
0.5		41.04 ± 0.18	18.32 ± 0.06	23.82 ± 0.04
1.0		41.09 ± 0.11	18.55 ± 0.04	23.41 ± 0.02
1.25		40.23 ± 0.09	17.78 ± 0.02	21.75 ± 0.01
1.5		40.90 ± 0.10	18.65 ± 0.04	22.91 ± 0.03
0 (control)	48	37.08 ± 0.13	11.31 ± 0.21	16.28 ± 0.07
0.25		38.79 ± 0.10	14.13 ± 0.08	16.08 ± 0.09
0.5		40.44 ± 0.07	17.59 ± 0.24	22.13 ± 0.11
1.0		40.85 ± 0.07	17.58 ± 0.04	22.61 ± 0.06
1.25		39.27 ± 0.08	16.61 ± 0.14	20.28 ± 0.06
1.5		39.50 ± 0.06	17.51 ± 0.16	21.13 ± 0.08
0 (control)	72	JS	JS	JS
0.25		JS	JS	JS
0.5		JS	JS	JS
1.0		38.91 ± 0.09	16.01 ± 0.10	20.83 ± 0.15
1.25		39.02 ± 0,17	16.20 ± 0.11	19.12 ± 0.49
1.5		39.09 ± 0.05	16.15 ± 0.04	19.96 ± 0.12

The results are expressed as a mean (n = 3) ± standard deviation from three independent experiments. JS—juice spoiled.

shows changes in colour parameters induced by the dissolution of lyophilised sumac in the carrot juice and the time of storage of the products.

The juices enriched with the lyophilised sumac extract exhibited slightly higher brightness, as indicated by the increased L* parameter, in comparison to the control samples. Moreover, the juices enriched with LSE were redder and yellower (increase in a* and b* parameter values) than the control sample (Table 3). The effect of sumac additives on the colour of food products was confirmed by Al-Marazeeq et al. [63]. In their study, the authors added sumac water extracts (concentrations: 0.5%, 1%, 1.5%, 3%, and 5% by weight) to wheat pan bread. The extracts were used to prepare dough, which was compared with control dough prepared with water only. The L* and b* parameters of the crust and crumb decreased significantly (p < 0.05), while the a* parameter describing the degree of redness increased.

3.3. Bioactive Compounds

Vitamin C (L-ascorbic acid) has many functions in the body: it is one of the most important antioxidants, ensures normal functioning of many enzymes, and takes part in collagen synthesis [64,65,66]. The content of this compound is therefore an important indicator of carrot juice quality. The vitamin C content in the analysed juices ranged from 3.07 to 4.24 mg/100 g (

Table 4. Contents of vitamin C, carotenoids, and polyphenols in carrot juice stored for 24, 48, and 72 h.

Storage Time	LSE Addition (g/100 mL)	Vitamin C (mg/100 g)	TCC (mg/100 g)	TPC (mg/100 mL)
24 h	0 (control)	3.34 ± 0.40	14.08 ± 0.08	20.33 ± 0.04
	0.25	3.25 ± 0.19	13.87 ± 0.13	25.86 ± 0.22
	0.5	3.73 ± 0.21	17.71 ± 0.14	46.33 ± 0.09
	1.0	4.00 ± 0.36	17.62 ± 0.02	47.15 ± 0.04
	1.25	4.08 ± 0.01	17.77 ± 0.08	50.62 ± 0.17
	1.5	4.24 ± 0.45	17.97 ± 0.08	55.38 ± 0.39
48 h	0 (control)	3.36 ± 0.16	13.40 ± 0.04	16.83 ± 0.39
	0.25	3.07 ± 0.09	13.82 ± 0.02	19.50 ± 0.87
	0.5	3.44 ± 0.17	17.71 ± 0.09	22.08 ± 0.09
	1.0	3.79 ± 0.40	17.41 ± 0.05	35.25 ± 0.30
	1.25	3.80 ± 0.35	17.28 ± 0.09	49.40 ± 0.26
	1.5	4.12 ± 0.20	17.36 ± 0.06	51.33 ± 0.22
72 h	0 (control)	JS	JS	JS
	0.25	JS	JS	JS
	0.5	JS	JS	JS
	1.0	3.52 ± 0.37	18.04 ± 0.02	37.12 ± 0.52
	1.25	3.32 ± 0.39	18.06 ± 0.02	49.67 ± 0.04
	1.5	3.55 ± 0.06	18.41 ± 0.06	56.61 ± 0.22

The results are expressed as a mean (n = 3) ± standard deviation from three independent experiments. JS—juice spoiled.

). The content of ascorbic acid in carrot juices is in a fairly wide range and may vary from 1.6 mg/100 mL [67] to 14.48 mg/100 mL [68]. Michalczyk et al. [69] reported that the content of this bioactive component in heat-untreated fresh carrot juice was 1.1 mg/100 g. In the control sample and carrot juices with lower proportions of LSE (0.25 to 0.5 g/100 mL), no determination of vitamin C was carried out after 72 h of storage, as these juices showed signs of spoilage (delamination, mucilaginous consistency, unpleasant odour). After 72 h of storage of the carrot juices, the vitamin C losses were 12–18%, compared to samples tested after 24 h of storage. The LSE addition increased the ascorbic acid content in the carrot juices, but a statistically significant difference compared to the control sample was observed in the case of the juice containing 1.5 g/100 mL LSE (Table 1).

Carotenoids are a group of compounds that not only give an appetising appearance to vegetables or fruit but are also important for human health through their antioxidant properties and participation in vitamin A production. In the tested juices, the average TCC ranged from 13.40 to 18.41 mg/100 g (Table 4). The LSE exerted an influence on the value of this parameter (Table 1). The addition of LSE in the amount of 0.5 g/100 mL increased the content of the analysed compounds by about 22%.

The slight decrease in the pigment content in the control samples is most likely related to many biochemical processes that carotenoids undergo during storage, first of all, the oxidation of unsaturated chains via photo-oxidation or autooxidation [70]. Anthocyanins, carotenoids and their derivatives, and phenolic compounds present in sumac are the dominant antioxidants [71,72] that can inhibit the formation of oxidation products in beverages. Hosseini et al. [73] reported a significant amount of β-carotene in oil extracted from sumac fruit (Rhus coriaria L.). Hence, its presence in the spice added to the carrot juice probably increased the total carotenoid content. In addition, it is likely that the slight acidification of the juice with LSE increased the content of the analysed compounds. As observed by Bell et al. [70] in their analyses of carrot juices with modified acidity, neutral and slightly alkaline conditions (pH 8 and 7) reduced the content of total carotenoids, while acidic conditions (pH 6–3) favoured an increase in their amount in carrot juice, which was due to an increase in the solubility of the pigments present in the vacuoles.

As shown in our study, the freshly pressed carrot juice was characterised by an average level of total polyphenols of 20.33 mg/100 mL after 24 h of storage (Table 4). Even small amounts of LSE added to the juice (0.5 g/100 mL) resulted in a significant increase (Table 1) in polyphenols (by about 56% compared to the control juice). Juices supplemented with 1.5 g of LSE had the highest content of polyphenolic compounds at an average level of 55.38 mg/100 mL after 24 h of storage. The additive used not only ensured the microbiological safety of the products but also enriched the juice with biologically active compounds, whose amount after 72 h was similar to the initial values. The increase in the content of polyphenolic compounds may be associated with reactions between oxidised polyphenols and the formation of new antioxidant compounds during juice storage [74] or the formation of compounds that react with the Folin–Ciocalteu reagent [75].

Rhus coriaria sumac fruit has high amounts of polyphenols, such as gallic acid, hydrolysed tannins, anthocyanins, and flavones [32,76,77]. In the present study, the extraction was carried out at 40 °C for 3 h, based on preliminary tests and previously published reports indicating effective recovery of bioactives in comparable conditions. Nonetheless, it is acknowledged that extended or sequential extraction protocols may further improve both the yield and the diversity of phytochemicals recovered. This issue warrants further investigation. Sumac has also been successfully used to enrich various types of bread with phenolic compounds [78,79] and to enhance the quality and oxidative stability of animal-derived products, such as cheese and ground beef [31].

3.4. Antioxidant Activity

The ABTS and DPPH radical scavenging activity assays and the FRAP method are commonly used to determine the antioxidant activity of plant compounds and their derivatives [79]. The antiradical potential measured by the DPPH assay observed in the sumac extracts is within a spectrum ranging from 863.70  ±  29.61 mmol TE/g to 2354.79  ±  37.07 mmol TE/g, depending on the solvent type [79]. On the other hand, the available data regarding carrot juice were obtained in the past years and probably do not fully reflect the current state of knowledge, mainly due to the introduction of new cultivars of carrot. In our study, the pure carrot juice had the lowest antioxidant activity, while the addition of freeze-dried sumac increased this parameter over tenfold. For example, in the DPPH test, it was 7.50 and 77.63 mmol TE/100 mL in the control and the sample with the highest addition of LSE, respectively, after 24 h of storage (

Table 5. Antioxidant activity of carrot juice stored for 24, 48, and 72 h.

Storage Time	LSE Addition (g/100 mL)	DPPH	ABTS	FRAP
		(mmol TE/100 mL)
24 h	0 (control)	7.50 ± 1.15	30.50 ± 0.77	130.74 ± 3.62
	0.25	11.79 ± 1.15	33.86 ± 0.88	149.49 ± 2.01
	0.5	27.07 ± 0.86	44.26 ± 0.55	344.38 ± 4.42
	1.0	53.67 ± 0.43	74.54 ± 0.44	462.84 ± 3.21
	1.25	66.72 ± 1.30	90.50 ± 0.44	751.19 ± 2.81
	1.5	77.63 ± 1.15	101.45 ± 1.33	994.66 ± 3.21
48 h	0 (control)	9.69 ± 0.92	24.65 ± 0.05	128.75 ± 4.82
	0.25	11.08 ± 0.52	24.81 ± 1.35	148.35 ± 2.01
	0.5	20.82 ± 0.13	34.37 ± 1.58	221.93 ± 4.26
	1.0	29.06 ± 1.05	41.14 ± 1.01	311.42 ± 2.81
	1.25	51.49 ± 1.05	68.78 ± 0.22	492.67 ± 2.81
	1.5	60.39 ± 1.05	85.75 ± 1.46	643.52 ± 0.80
72 h	0 (control)	JS	JS	JS
	0.25	JS	JS	JS
	0.5	JS	JS	JS
	1.0	32.32 ± 0.52	63.49 ± 1.17	365.11 ± 8.84
	1.25	44.71 ± 0.69	84.77 ± 1.05	529.32 ± 5.62
	1.5	53.51 ± 0.64	99.35 ± 1.05	583.58 ± 6.03

The results are expressed as a mean (n = 3) ± standard deviation from three independent experiments. JS—juice spoiled; TE—Trolox Equivalent.

). Interestingly, the activity measured by the ABTS assay during storage increased between the second and third day due to the increase in the polyphenol content.

The level of scavenging of ABTS•+ by the studied sumac extracts was higher than that of DPPH· radicals, probably because ABTS•+ cation radicals are more sensitive to high-molecular-weight phenolics and more reactive than DPPH· radicals, and the reaction of ABTS•+ radicals with an antioxidant is completed within 1 min [80].

The correlation analysis confirmed that the antioxidant activity was most strongly associated with the content of phenolic compounds and, to a slightly lesser extent, with vitamin C (

Table 6. Correlations between the content of bioactive ingredients and the antioxidant activity of carrot juices.

	Vitamin C	TCC	TPC
FRAP	0.806	0.696	0.878
DPPH	0.842	0.742	0.912
ABTS	0.679	0.745	0.928

). In contrast, the carotenoid levels had the weakest influence on antioxidant capacity and reducing power measured by the FRAP assay. Similar findings were reported in studies on Rhus coriaria extracts by other authors. Kosar et al. [40] demonstrated that all tested extracts (ethyl acetate and hydrolysed) and their respective fractions exhibited significant DPPH radical scavenging activity at a concentration of 30 µg/mL. A strong positive correlation (R² = 0.878) was observed between total phenolic content and antioxidant activity, indicating that phenolics are the main contributors to antioxidant capacity. Vitamin C also contributed, but to a lesser extent. The carotenoid levels were low and had a minimal impact on antioxidant and reducing activity. The reducing power measured by the FRAP assay followed a similar trend, correlating strongly with the phenolic content [41].

It is very clear that the addition of the LSE contributed significantly to the increase in the activity measured by the ABTS and DPPH assays and the reducing capacity measured by FRAP. In terms of the level of supplementation, the LSE addition from the concentration of 1 mg/100 mL of juice was the most effective. This was evidenced by both the statistical analysis (Table 1) and the fact that samples below this limit were spoiled on the third day. It is difficult to compare the present results with the literature because the majority of studies focus on alcoholic extracts, which are often subjected to additional processing, such as defatting. Interestingly, a comparison of sumac extracts conducted by Bursal and Köksal [41] revealed that the antioxidant capacity, radical scavenging, and reducing power potential of the water extract were markedly higher than those of the ethanol extract. However, it should be noted that a limitation of this study was the fact that the extracts were not subjected to precisely the same sample preparation procedures. Specifically, the water extracts underwent lyophilisation, while the ethanol extracts were evaporated.

3.5. Phenolic Compound Profile—LC-MS

The juices were also subjected to detailed analysis of the content of individual phenolic ingredients.

Table 7. Presence of bioactive compounds in juices during storage.

Component	LSE Addition (g/100 mL)
	24 h						72 h
	0	0.25	0.5	1	1.25	1.5	1	1.25	1.5
2,3-Dihydroybenzoic acid		+	+	+	+	+	+	+	+
4-Hydroxycoumarin		+	+	+	+	+	+	+	+
Anhydro-secoisolariciresinol							+	+	+
Caffeic acid	+	+	+	+	+	+	+	+	+
Catechin					+	+	+	+	+
Catechol							+	+	+
Chlorogenic acid	+	+	+	+	+	+	+	+	+
Coumarin				+	+	+	+	+	+
Ferulic acid	+	+	+	+	+	+	+	+	+
Gallic acid				+	+	+	+	+	+
Isoferulic acid		+	+	+	+	+	+	+	+
Isoquercetin	+	+	+	+	+	+	+	+	+
Myricetin				+	+	+	+	+	+
Phloroglucinol	+	+	+	+	+	+	+	+	+
Protocatechuic acid				+	+	+	+	+	+
Pyrogallol	+	+	+	+	+	+	+	+	+
Quercetin	+	+	+	+	+	+	+	+	+
Quercetin 3′-O-glucuronide				+	+	+		+	+
Quercetin3-O-glucosylxyloside			+	+	+	+	+	+	+
Rosmanol							+	+	+
Syringic acid				+	+	+	+	+	+
trans Ferulic acid		+	+	+	+	+	+	+	+
Tyrosol							+	+	+
Vanillic acid	+	+	+	+	+	+	+	+	+
Vanillin				+	+	+	+	+	+

presents the results of the LC-MS analyses of beverage samples after 24 and 72 h, as no changes were observed after 48 h, compared to 24 h. Various groups of bioactive compounds were detected: chalcones, flavonols, flavones, and phenolic acids.

Several phenolic compounds were detected in all the analysed carrot juice samples, including the control, indicating that the carrot juice itself was a natural source of these constituents. The following compounds were identified: 6-Hydroxyluteolin7-O-rhamnoside, Apigenin7-O-apiosyl-glucoside, Glycitein 7-O-glucoside, Isorhoifolin, quercetin and its derivatives, Phloroglucinol, Pyrogallol, scopoletin, acids (Caffeic acid, Chlorogenic acid, Ferulic acid, Vanillic acid), and their derivatives (Ferulic acid 4-O-glucoside). In contrast, certain bioactive constituents were only detected in samples supplemented with the lyophilised sumac extract (LSE), indicating that these compounds originated from the additive. They were represented by, e.g., 3′-Hydroxygenistein, 4-Hydroxycoumarin, Coumarin, Myricetin, Vanilline, Catechin, Apigenin-7-O-glucoside. Moreover, several acids known to be abundant in sumac, such as gallic acid, isoferulic acid, trans ferulic acid, Protocatechuic acid, 2,3-Dihydroxybenzoic acid, and syringic acid. Notably, such compounds as anhydro-secoisolariciresinol, catechol, rosmanol, and tyrosol were identified only in the LSE-supplemented carrot juices after 72 h of storage, suggesting potential transformations during storage. These findings were corroborated by the spectrophotometric analyses, which revealed an increase in the total phenolic content on the third day of storage, further supporting the hypothesis that new antioxidant compounds may form over time due to interactions with sumac-derived components or enzymatic/microbial activity.

Our results are consistent with those obtained by other authors. Przybylska et al. [42] analysed carrot roots and juices and identified the presence of various chalcones, flavonoids, and phenolic acids. Chlorogenic acid, two dihydrochalcones (phloridzin and phloretin), caffeic acid, and quercetin were detected as the main compounds. As shown in the literature, sumac water extract ensures the addition of such compounds as vanillic acid, gallic acid, myricetin, ellagic acid, fumaric acid, and resveratrol [81]. Other authors analysed various extracts, and the results were similar. The content of both phenolic and flavonoid substances in the ethanol extract was approximately ten times higher than that in the aqueous extract. Nine compounds were identified in the ethanolic extract of sumac, with substantial quantities of fumaric acid (452.78 mg/kg), pyrogallol (123.28 mg/kg), and gallic acid (86.77 mg/kg). The water extract of sumac comprised eight compounds, primarily fumaric acid (180.72 mg/kg), epicatechin (21.2 mg/kg), and gallic acid (19.31 mg/kg). Furthermore, kaempferol-3-O-rutinoside (10.16 mg/kg) and ellagic acid (12.29 mg/kg) were exclusively found in the ethanolic extract, while rutin (0.49 mg/kg) was present only in the water extract of sumac [82]. Unfortunately, there are no results in the literature regarding juices with the addition of sumac or the impact of storage. Therefore, it is challenging to directly relate to our results showing the presence of new compounds after 72 h. It can be presumed that this may also result from the activity of bacteria, fungi, or yeasts. For instance, Romboli et al. [83] reported that the formation of tyrosol and hydroxytyrosol during fermentation was influenced by yeast strains and oxygen availability. Furthermore, endogenous plant enzymes in carrot or sumac tissue can significantly alter phenolic profiles during storage. Polyphenol oxidase (PPO) is a primary culprit: it hydroxylates monophenols and oxidises o-diphenols to o-quinones. Thus, PPO-mediated oxidation depletes certain simple phenols and generates new polymeric phenolic species. In practice, fresh carrot-sumac juice darkens as plant phenols, PPO, and residual oxygen react [84]. Storage-related chemistry is further complicated by reactions between polyphenols and other matrix constituents. Non-enzymatic browning (Maillard-type) reactions between reducing sugars and amino compounds are well-known in juices [85]. Taken together, these processes often yield new high-molecular-weight phenolic compounds during storage. In summary, the additional phenolic compounds observed after 72 h can plausibly arise from enzymatic oxidation, microbial metabolism, and non-enzymatic condensation reactions that jointly reshape the phenolic chemistry of juice. It should be noted that, although this study focused on the overall antioxidant potential and general bioactive content, future research could include the targeted quantification of individual compounds to provide a more detailed understanding of their specific contributions to biological activity.

4. Conclusions

Consumers increasingly demand food that is not only nutritionally adequate but, above all, microbiologically safe. Freshly pressed, heat-untreated juices are rich in health-promoting ingredients but are also highly susceptible to spoilage. Consequently, they pose a potential risk to human health and can lead to economic losses for producers. To extend the shelf life of such products, natural-origin additives are used more frequently. Unlike many synthetic substances, they typically do not exert toxic effects.

Our results indicate that lyophilised sumac extract (LSE) dissolved in carrot juice exhibits strong antimicrobial properties by inhibiting the growth of mesophilic aerobic bacteria, yeasts, and moulds. Notably, the addition of LSE was effective in limiting microbial growth, even when the initial microbial load in the product was relatively high. Furthermore, the extract enriched the juice with biologically active compounds—particularly polyphenols and carotenoids—and enhanced its total antioxidant capacity and reduction potential. Carrot juices supplemented with 1.5 g/100 mL of LSE maintained their quality during refrigerated storage (6 °C) for up to 72 h.

Given its multifunctional benefits, LSE represents a promising ingredient for the development of “clean label” juice products with extended shelf life and enhanced functional properties. As health-conscious consumers are increasingly seeking naturally fortified foods, the use of sumac extracts could not only support innovative product development but also promote the cultivation and economic utilisation of sumac in its traditional growing regions.